CHAPTER 5 PHYTOCHEMICAL STUDIES OF CYCLEA PELTATA

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1 CHAPTER 5 PHYTOCHEMICAL STUDIES OF CYCLEA PELTATA

2 Aim: To investigate the phytochemical compounds of C. peltata extracts (Hexane, Chloroform, Methanol, Crude and Alkaloid extracts) 5.1. Introduction Plants generally contain primary and secondary metabolites namely alkaloids, terpenoids, flavonoids, saponins, coumarins, glycosides, phenolics, carboxylic acids, aminoacids, sugars, proteins etc. These phyto-constituents impart the specific characteristics and properties of plants. Therefore, it is obligatory to resolve all of the phytochemical constituents present in the plants in order to ensure the consistency and repeatability of pharmacological, antimicrobial and clinical research, to understand their bioactivities, identify the active principles (components) and possible side effects of active compounds Plants are the resource of primary and secondary metabolites namely alkaloids, terpinoids, flavanoids, saponins, coumarins, glycosides, phenolics, carboxylic acids, aminoacods, sugars, proteins etc. these phytochemicals have significant biological functions and also which contribute specific characteristic and property of the plant. The therapuuetic use of the plant is due to presence of these compounds. Hence the study and charecterisation of such phytocompounds have great significant in pharmacological, antimicrobial and clinical research. Different methods are availabile for separation, purification, identification and charectirisation of such phytoconstituents present in plants. Ideally, fresh plant tissue should be used for phytochemical analysis. Alternatively plants may be dried before extraction. It is essential that the drying operation is carried out under controlled conditions to avoid too many chemical changes occurring. It should be dried as quickly as possible without using high temperature. The botanical identity of the plant studied must be authenticated by an acknowledged authority at the beginning stage in the investigation. The precise mode of extraction naturally depends on the texture and water content of the plant material being extracted and on the type of substance that is being isolated. Alcohol, in any case, is a good all-purpose solvent for preliminary extraction. The classical chemical procedure for obtaining organic constituents from dried plant tissue is to continuously extract powder material in a Soxhlet apparatus with a range of solvents starting in turn with ether, petroleum and 69

3 chloroform and then using alcohol and ethyl acetate. However, one rarely achieves complete separation of constituents and the same compounds may be recovered in several fractions. Quantitative and qualitative estimation of these phytocostituents carried out using technique such as chromatographic and spectrophotometric methods. Chromatographic techniques are the most useful and popular method among this High performance thin layer chromatography is effective and powerful tool for linking the chemical constuents profile of the plant extracts (Yamunadevi et al., 2011; Bobby et al., 2012). Identifying a plant constituent, once it has been isolated and purified, it is necessary first to determine the class of compound and then to find out which particular substance it is. Complete identification include melting point (Solid), boiling point (liquid), optical rotation and R f. Spectral characteristics like ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR) and mass spectral (MS) measurements are also highly informative Materials and Methods Plant material and preparation of the extract The procedure is mentioned in Materials and Methods (4.1.1.) Preliminary Phytochemical Screening The different extracts of C. peltata (CP, HCP, CCP, MCP and ACP) were analysed for the presence of secondary metabolites as per the method described by Harborne (1984) Test for carbohydrates/glycosides: (Molish test) 5 mg of extract was mixed with 0.5 ml of distilled water and added 2 drops of 10% β naphthol in ethanol or choloroform.to this mixture, added 1 ml of concentrated H 2 SO 4 slowly through the sides of the test tube so that it forms a ring at the middle of the two solutions. The formation of a violet ring indicated the presence of carbohydrate/glycosides. 70

4 Test for Alkaloids: Dragendroff 's test: Dragendroff s reagent was prepared as follows: Dissolved 0.6 gm of bismuth subnitrate in 2 ml of concentrated HCl and added 10 ml of distilled water-stock solution- A. Dissolve 6 gm of KI in 10 ml of water-stock solution B. Solution A and solution B were mixed together with 7 ml of concentrated HCl and 15ml distilled water and the whole solution was diluted with distilled water. Most of the alkaloids gave an orange red precipitate with the reagent Wagners' Test: Wagner's reagent was prepared as, 1.72 gm of Iodine and 2gm of KI were dissolved in 5 ml of distilled water and the solution was made up to 100 ml with distilled water. Many alkaloids gave a brown flocculent precipitate with Wagner's reagent Mayer's Test: 1.36gm of HgCl 2 was dissolved in 60 ml of distilled water stock solution A, 5 gm of KI was dissolved in 10 ml of distilled water-stock solution B. Both the solutions were mixed and diluted to 100 ml with distilled water. Since the reagent reacted only with salts of alkaloids, the alkaloidal solution was made distinctly acidic with HCl or H 2 SO 4 before adding the reagent. The reagent was added drop wise. A white precipitate indicated the presence of alkaloids Test for Saponins: A little of the extract was shaken well with water. The formation of froth in the test tube which persisted for a few minutes showed the presence of saponins Test for Steroids/Terpenoids: The extract was dissolved in dry CHCl 3, to this added 2 ml of acetic anhydride, mixed well. Added 2 ml of concentrated H 2 SO 4 through the sides of the tube. A green colour indicated the 71

5 presence of steroids and pink colour indicates the presence of terpenoids (Liebermann-Burchard reaction) Test for Tannins 5gm plant powder was boiled in100 ml water for 5 mins. The extract was filtered and to 5 ml clear filtrate 2 ml of 2% gelatin solution is added. A curdy white precipitate indicated the presence of tannins Test for Flavanoids (Shinoda s test) Extract was dissolved in methanol and magnesium turnings were added followed by concentrated HCl, drop by drop. Pink colour indicated the presence of flavanoids Test for Coumarins: Extract was dissolved in methanol and alcoholic NaOH was added. A yellow colour that appeared while adding drops of concentrated HCl indicated the presence of coumarins Estimation of total alkaloids in CP (Sreevidya and Mehrotra, 2003) 15 gm of plant material was extracted with 200ml chloroform: ether: alcohol (90%; 23: 8: 2.5) for 10 mins. To this 6 ml dilute ammonia was added and the flask was shaken for 1 h and allowed to stand for 8 h with occasional shaking. To this, 10ml water was added and shaken vigorously. When the drug has settled 100 ml of the solution was drawn out and filtered into a separator and washed with a few ml of mixture of solvent ether and chloroform, shaken with 0.5N sulphuric acid for complete extraction of alkaloids. The combined acid extract was filtered and made alkaline with dilute ammonia solution. The alkaloids were liberated with chloroform and washed with chloroform. The chloroform was removed completely using vacuum evaporator and dried completely in desiccator. 5ml of alcohol was added and concentrated using vacuum evaporator and dried completely in desiccator. The evaporation with alcohol was repeated and residue dried to constant weight in vacuum evaporator as total alkaloids. 72

6 Estimation of total phenolics in CP (Singleton et al., 1965). The total phenolic content in CP was determined by using Folin Ciocalteu s method (Singleton et al., 1965). A standard curve was prepared using different concentrations of gallic acid (10, 25, 50, 100, 110, 125, 150 and 200 µg/ml) prepared in ethanol. To the above standard solutions of gallic acid (1 ml each), 5 ml of Folin Ciocalteu s reagent (1:10 dilution in distilled water) was added and after 8 min, 4 ml of 7.5% sodium carbonate was added. These solutions were incubated at room temperature for 2 h and their absorbances at 765 nm were measured on UV-Visible spectrometer (UV-1650PC, Shimadzu, Japan). For the test solution, 1 ml of CP (10 mg in 10 ml EtOH) was mixed with 5 ml of Folin Ciocalteu s reagent and 4 ml of 7.5% sodium carbonate was added after 8 min and incubated at room temperature for 2 h and its absorbance was also read at 765 nm. The gallic acid equivalent (GAE) was plotted and the total phenolic content of CP was calculated in terms of Gallic acid equivalents by considering the dilution factor Thin Layer Chromatography (TLC) of the C. peltata extracts TLC of the extract was performed on a pre-coated silica gel plate purchased from Merck India Ltd using solvent system toluene: ethyl acetate: diethylamine (7.2: 2: 0.8). The Rf value of the different spots were calculated Coloumn Chromatography of ACP and isolation of tetrandrine (TET) ACP (1.6 gm) is subjected to column chromatography over neutral Alumina (50 gm) using toluene: ethyl acetate: diethylamine (6.9: 3: 0.1) isocratic elution. 10 ml fractions were collected and fractions 6-8 gave pure compound (9.4 mg) Characterization of isolated compound Ultraviolet (UV) spectrum and Thin Layer Chromatography (TLC) of isolated compound The UV spectrum of the isolated compound has been carried out at 209 nm using the instrument. TLC of the compound using standard tetrandrine has been carried out using solvent system toluene: ethyl acetate: diethylamine (7.2: 2: 0.8). The plates were evaluated under UV 73

7 (209nm) and sprayed with Dragendroff s reagent, the developed spots were evaluated. The Rf value of the different spots were calculated Direct analysis in real time- mass spectrometry (DART-MS) of alkaloid extract (ACP) (Nilles et al., 2009; Jana et al., 2011). The mass spectrometer used was a JMS-T100 LC (Accu ToF) atmospheric pressure ionization time-of-flight mass spectrometer (Jeol, Tokyo, Japan) fitted with a DART ion source. The mass spectrometer was operated in positive-ion mode. The DART ion source was operated with helium gas flowing at 4.0 L/min. The gas heater was set to 300 C. The potential on the discharge needle electrode of the DART source was set to 3000 V. Orifice 1 potential was set at 28V. The ACP extract was positioned in the gap between the DART source and mass spectrometer for measurements. Data acquisition was from m/z 10 to LCMS/MS of alkaloid extract LCMS/MS analysis of ACP extract has been carried out using the instrument Estimation of Tetrandrine (TET) using High Performance Thin Layer Chromatography (HPTLC) (Modified method of Shine et al., 2009) HCP, CCP, MCP, CP and ACP were subjected to HPTLC analysis (Camag, Switzerland), using pre-coated TLC plate of silica gel 60 F 254 (Merck, India). Extracts and different amounts of TET were applied using Linomat V. TLC was developed and then scanned densitometrically at 209 nm using CAMAG TLC Scanner Preparation of Extracts: HCP, CCP, MCP, CP and ACP were prepared (5mg/ml concentration each) and applied as 40µl HCP, 10µl CCP, 10µl MCP, 2.5µl ACP and 10 µl CP respectively on precoated TLC plate using Linomat V. Developed the plate in the solvent system (Toluene: Ethyl acetate: Diethyamine; 7.2: 2: 0.8) in a twin trough chamber to a distance of 8 cm, dried and scanned the plate densitometrically at 209 nm using CAMAG TLC Scanner 3 and recorded the peak area of standard tetrandrine applied. Calculated the amount of tetrandrine present in the extracts from calibration curve given below. 74

8 Preparation of standard tetrandrine and calibration curve Standard solution was prepared in mg/ml concentration and applied 0.5µl, 1.0 µl, 1.5 µl, 2.0 µl, 2.5 µl, 3.0µl and 3.5 µl of standard solution so that the tracks contain 0.5µg, 1.0 µg, 1.5 µg, 2.0 µg, 2.5 µg, 3.0µg and 3.5 µg of tetrandrine respectively on precoated TLC plate using Linomat V. Developed the plate in the solvent system (Toluene: Ethyl acetate: Diethyamine; 7.2: 2: 0.8) in a twin trough chamber to a distance of 8 cm, dried and scanned the plate densitometrically at 209 nm using CAMAG TLC Scanner 3 and recorded the peak area of standard tetrandrine applied Results Preliminary Phytochemical Screening The result showed the presence of alkaloid, saponin, steroid and terpinoid in ACP, CCP and CP (Table. 6) Estimation of total alkaloids in CP Total alkaloid in C. peltata was estimated as 207mg/g UV Spectrum of tetrandrine The UV spectrum of the isolated tetrandrine is shown in Fig. 11.a Estimation of Tetrandrine (TET) using High Performance Thin Layer Chromatography (HPTLC). HPTLC estimation, ACP has got the highest content of tetandrine µg/mg and followed by CP 29.62µg/mg, CCP 23.46µg/mg, MCP 18.82µg/mg and HCP 1.25µg/mg (Table. 7) Direct Analysis in Real Time (DART) analysis of alkaloid extract (ACP) Representative DART-MS spectra of the alkaloid extract of C. peltata is shown in Fig. 11.b. In DART-MS data each peak represents the molecular weight (MW) + 1 (M+1) value. DART analysis detect the presence of bisbenzyl isoquinoline alkaloids, Tetrandrine (TET., C 38 H 42 O 8 N 2 ; MW ), fangchinoline (FAN., C 37 H 40 N 2 O 6 ; MW ) and coclaurine 75

9 (CoCl., C 17 H 19 NO 3 ; MW ) which were detected as peaks of M+1 values (TET), (FAN) and (CoCl) respectively LCMS of alkaloid extract (ACP) The major masses in the LCMS spectrum (Fig. 11.c) detected were M+1 values of TET (623.3), FAG (609.2) and other unknown (413.3) and also the small peak may be the M+1 value of bisbenzylisoquinoline alkaloid curine (594.27) Discussion Preliminary phytochemical studies showed presence of triterpenoids, phytosterols, alkaloids and saponins. Alkaloid is present in all the extracts including hexane extract. Family Menispermacea is a rich source of alkaloids. C. peltata roots are reported to contain the alkaloids d-tetrandrine and dl-tetrandrine and fangchinoline (Rastogi and Mehrotra 1999; Reddi et al., 2005). Based on the cited literature, the Indian sample of the C. peltata yielded tetrandrine as the major alkaloid (Anonymous, 2004). HPTLC studies showed tetrandrine is present in all the extracts including hexane extract. Alkaloid extract (ACP) has got the highest TET content than other extracts. CP had higher TET content than CCP and MCP respectively HCP had very low TET content when compared to other extracts (Fig. 2-10). TET has been isolated from the root of C. peltata using column chromatographic technique and the compound has been confirmed using Co-TLC with authentic TET and also from UV spectrum of the compound (Figs. 11.a). DART and LCMS analysis also confirmed the TET (C 38 H 42 O 8 N 2 ) with molecular weight From both the data it is clear that fangchinoline (C 37 H 40 N 2 O 6 ) is present in the root of C. peltata with molecular weight which is in line with the previous report (Rastogi and Mehrotra 1999; Reddi et al., 2005) (Figs. 11.b-c). Coclaurine and curine were detected in DART and LCMS respectively. TET in C. peltata is reported to inhibit TNF-α, COX-2 (Ferrante et al., 1990), nuclear factor Kappa B (NF κb) (Hsu et al., 2007), transforming growth factor-beta1 (TGF-β1), inhibits COX-2 expression without inhibiting the COX-1 levels (Wu and Ng, 2007), free radical scavenging (Cao, 1996), antifibrotic effect (Hsu et al., 2007; Park et al., 2000), inhibit Ca 2+ transmembrane movement (Huang, 1999). It is more effective for cirrhotic patients with portal hypertension in preventing recurrent variceal bleeding (Li et al., 1995) induces apoptosis of HSC 76

10 cells (Zhao et al., 2004). Fangchinoline is known to inhibit Ca 2+ transmembrane movement and histamine release (Nakamura et al., 1992). Coclaurine has a potent antispasmodic activity, related to an inhibiting effect of extracellular calcium, an intracellular effect. Table. 6. Preliminary phytochemical analysis Extracts Glycosides Alkaloids Saponins Steroids Terpenoids Tannins Flavanoids Coumarins HCP CCP MCP CP ACP Table. 7. Estimation of Tetrandrine (TET) using HPTLC Track Sample/standard Amount applied (µg) Area Amount Calculated (µg/mg) 1 Standard Standard Standard Standard Standard Standard Standard Hexane CP (HCP) Chloroform CP (CCP) Methanol CP (MCP) Alkaloid CP (ACP) Crude CP (CP)

CHAPTER 5 PHYTOCHEMICAL EVALUATION

76 CHAPTER 5 PHYTOCHEMICAL EVALUATION Phytochemistry is mainly concerned with enormous varieties of secondary plant metabolites which are biosynthesized by plants. The plant kingdom represents a treasure